Professional Documents
Culture Documents
Vibrionaceae
The name “vibrio” is derived from
characteristic vibratory motility.
(vibrare= to vibrate)
General charcters of Vibrionaceae
• 1884: Rediscovers
Vibrio cholerae
BACKGROUND
Cholera, is a Greek word, which means the
gutter of the roof. It is caused by bacteria:
Vibrio cholerae, which was discovered in 1884
by Robert Koch during a diarrheal outbreak in
Egypt.
The genus as 40 species to which 12 are
responsible for intestinal and extra intestinal
infections in man. Most commonly isolated from
clinical specimens are- V. cholerae, V.
parahemolyticus, V. vulnificus, V. mimicus & V.
alginoliticus.
V. CHOLERAE
V. cholerae has 2 major biotypes: classical
and El Tor, which was first isolated in Egypt in
1905. Currently, El Tor is the predominant
cholera pathogen worldwide.
The organism is a comma-shaped, gram-
negative, short, curved, cylindrical rods, aerobic
bacillus whose size varies from 1-3 um in length
by 0.5-0.8 um, with rounded or pointed ends
It is actively motile with a single polar
flagellum. The motility is of the darting type
under a microscope suggest a ‘swarm of gnats’
(swarm= group, Gnat= winged insects- flea,
mosq).
Vibrio cholerae (gram stain)
PLATING MEDIA
1. Alkaline bile salt agar (BSA)-ph-8.2 is a
simple media, widely used, colonies are
similar to those on nutrient agar.
2. Monsur’s gelatin taurocholate trypticase
tellurite agar (G.T.T.A)-ph-8.5, useful for
cholera and other vibrios isolation from stool.
After 24 hours colonies-small 1-2 mm in
diameter, translucent, with greyish black
centre and a turbid halo around due to
hydrolysis & denaturation of gelatin.
• Thiosulphate citrate bile salt sucrose agar (T.C.B.S.)
(pH-8.6)- mostly used as selective media,
commercially available and widely used media.
Bromothymol blue is used as an indicator . The
colonies of V. cholerae are yellow due to sucrose
fermentation and blue-green of V. parahemolyticus
due to non- sucrose fermenters.
Identification
• 1-STRING TEST
• 2-IMViC Reaction
• 3-Nitrate reduction- +ve
• 4-Catalase+ve,Oxidase+ve,Urease-ve
• 5-Decarboxylase test-Ly+ve, Or+ve, Arg-ve
• 6-Sugar fermentation-glu+ve, suc+ve,
manitol+ve, mal+ve, manno+ve with acid &
no gas, Arb & Dul –ve
• String test- a loopful of growth is mixed
with a drop of 0.5% sodium deoxycholate
in saline on a slide. In positive test the
suspension loses it’s turbidity, becomes
mucoid and forms a “string” when the
loop is drawn slowly away the test is used
to separate vibrio species from Aeromonas
spp. & P. shigelloides (D/D).
Biochemical reactions
1.Carbohydrate metabolism is fermentative,
producing acid but no gas. Cholera vibrio
ferments glucose, mannitol, maltose, mannose
and sucrose.
2.Cholera-red reaction- V. cholerae is strongly
indole positive and reduces nitrates to nitrites,
when a few drops of concentrated sulphuric acid
is added to a 24 hrs peptone water culture- a
reddish pink color is developed due to formation
of nitroso- indole.
3. It is catalase & oxidase positive, MR & urease
negative.
4. Decarboxylates lysine & ornithine but
does not utilize arginine.
5. Gelatin is liquefied- funnel shaped or
“napi form” in 3 days at 220C.
6.Vogus-Proskauer reaction and hemolysis
of sheep RBC’s are positive in ElTor
biotype. Negative in classical biotype.
Resistance
They are killed-
• By heating at 56ºC for 30 min.,within few seconds
at boiling,quickly on dry fomites and in sewage
polluted water,in Gange’s water due to large
amount of vibriophages present,by disinfectants,
in few minutes on exposure to gastric juices.
They survives-
• For 1-2 weeks in clean, nonacid fresh or sea
water, few months in sterile sea water- survival in
nature,few days on moist foods, vegetables, fish and
cooked foods, for a week in refrigerator.
Antigens- Its antigenic structure consists of a
flagellar H antigen and a somatic O antigen.
It is the differentiation of the latter that
allows for separation into pathogenic and
nonpathogenic strains.
UP TO 200
NOW
NAG vibrios are mostly non – pathogenic,commensals
and isolated from the environment but can cause a
cholera like disease, agglutinated by their specific
antisera . The serogroup O139 identified in 1992 from
India (Chennai) causes epidemics of cholera and
possibly 8th pandemic. Therefore O1 and O139 are
responsible for epidemics and pandemics worldwide.
Biotypes of O1 V. Cholerae – 2 Biotypes differentiated
by biochemical and other test, agglutinated by O1
antisera.
Classical vibrio cholerae
Eltor vibrio cholerae
The Eltor vibrios were isolated by Gottschlich (1905)
from 6 Haj pilgrims at the Tor quarantine station on
the sinai penninsula (Egypt)
• These are identical except in biochemical and other
tests to classical cholera vibrios
SEROTYPES – based on minor surface antigenic
characters , both biotypes were classified further into
3 serotypes
Ogawa – agglutinated by its own antisera
Inaba – agglutinated by its own antisera
Hikojima – agglutinated by both antisera
• Strains of V.cholerae 0 1 may be further subdivided on
the basis of their O (A,B & C) antigens into 3 subtypes
– SEROTYPE O ANTIGENS
Ogawa AB
Inaba AC
Hikojima ABC
• V. cholerae O139 From Chennai it spreads to
rest of India, Bangladesh, South east Asia ,
China, Pakistan & parts of Europe and this may
be the start of 8th pandemic ? .
• It is non agglutinable to O1 and other available
antisera , therefore assigned to a new serotype
O139 Bengal, resembling to Eltor biotype
closely & replaced it gradually as well as coexist
in endemic areas.
• It is more invasive capsulated and causes
bacteremia
• O-1 strain vaccines could not protect against
O139 cholera
• It can be agglutinated by O139 antisera.
PHAGE TYPING
Strains of classical biotype of V.cholerae 01 can be
divided into 5 types by means of 3 phages (I-III) &
(IV) phage lyses all classical but not El Tor strains.
Phage type Sensitivity To Phase group
I II III IV
1 + + + +
2 _ + + +
3 + - + +
4 _ - + +
5 + + - +
PHAGE TYPING (ElTor)
• On basis of lysis of 4,phages ,El Tor
strains can be divided into 6 types . All
these strains are lysed by fifth (V) phage.
PHAGE SENSITIVITY TO PHASE GROUP
I II III IV V
1 + + + + +
2 + + + _ +
3 + + _ + +
4 + + _ _ +
5 + _ _ _ +
6 _ + _ _ +
VIBRIO II
• Cholera is an acute diarrheal disease caused by
cholera vibrio. In severe form, it presents as-
Profused diarrhea and
copious effortless vomiting
• This may lead to hypovolemic shock and death
in less than 24 hours.
• In treated cases the disease may last for 4-6
days, patient may pass a total volume of liquid
stool equal to twice his body weight. Therefore
cholera is a dramatic and terrifying illness.
• The incubation period < 24 hours- 5 days, the
symptoms are mild with ElTor biotype.
• The severity varies widely from rapidly fatal
disease to a transient asymptomatic colonization.
About 60% infections by classical vibrio & 75%
with ElTor remain asymptomatic.
• All the clinical feature of severe cholera results
from the massive loss of fluid & electrolytes.
• The stool is typically a colorless watery fluid with
flecks of mucous, resembling water in which rice
has been washed- rice water stools. The stool is
odorless, rich in bicarbonates, isotonic electrolyte
solution with little protein.
• It leads to decreased extracellular fluid volume,
hemoconcentration, hypokalemia, acidosis and
shock.
Complication are-
• Muscular cramps, renal failure, pulmonary
oedema, cardiac arrhythmias and paralytic ilius
i.e., profound dehydration, circulatory collapse
and anuria.
• Mortality- 25-50% in untreated cases.
Death attributable to:
Hypovolemic shock (due to abnormally low
volume of circulating fluid (plasma) in the body.
Metabolic acidosis (pH shifts toward acid side
due to loss of bicarbonate buffering capacity)
• ZIMBABWE: At a cholera
treatment center in the
town of Chegutu in
Mashonaland West
Province, a girl receives
intravenous fluids through a
drip into her arm.
Intravenous administration
of rehydration solution is
needed in severe cholera
cases to replace the fluids
lost through diarrhea and
vomiting. A large hole is cut
into her portable bed, with
a bucket placed
underneath, for patients
who are too weak to walk
to a proper latrine
• Above: A Cholera patient under intensive care at
HLSS' Owinykibul Army Barack Cholera Treatment
Centre, Eastern Equatoria State in 2014
History & Epidemiology
Cholera is an epidemic and endemic disease,
exclusively a human disease, spread by contaminated
food and vegetables by feces of carriers or patients.
Its homeland is large deltaic area of Ganges &
Brahmaputra in Bengal, where the disease is known
from ancient times. In Kolkata peak is in hot dry season
& ends with the onset of monsoon but extends to
neighboring states during rainy season.
Cholera is notifiable disease, about 30 lakhs cases
occur annually and 2 lakhs are reported to WHO,
resulting in more than 1 lakh deaths annually.
•Till in the early 19th century cholera was confined to India,
periodically causing large epidemics in different parts. From
1817-1923 cholera spread from Bengal in 6 separate
pandemics, involving most parts of the world.
About 8 pandemics occurred to date -
1. 1817-`23: First Pandemic
2. 1829-`51: Second Pandemic
3. 1852-`60: Third Pandemic (Pacini)
4. 1863-`79: Fourth Pandemic
5. 1881-`96: Fifth Pandemic (Koch).
6. 1899-`1923: Sixth Pandemic
7. 1961- still on: Seventh Pandemic
8. 1992-?: Eighth Pandemic
7th pandemic started for the first time outside India in
Celebes, Sulawesi (Indonesia) by another biotype ElTor.
In the 8th pandemic? Cholera returned to India, caused
by NAG O139 Bengal, discovered in Chennai.
Pathogenesis
Enters orally via contaminated water or food.
Vibrios are highly susceptible to acids in stomach
& small doses do not cause infection.
High infectious dose: >108 bacilli but much lower
dose 103 -105 bacilli with achlorhydria or
hypochlorhydria, meditation or surgery (partial
gastrectomy) predisposes to cholera.
The vibrio begin to multiply in alkaline
environment in jejunum, adheres with epithelial
cells with the help of enzymes, fimbria such as
“toxin corregulated pilus (TCP)” & colonize. Then
remain attach to epithelial surface but do not
damage or invade. The changes are biochemical
due to secretion of cholera toxin.
Cholera toxin- it is an enterotoxin, produced when vibrio
multiplies on epithelial cell surface. It is similar to heat
labile toxin (LT) of E. coli in structure, chemically,
antigenically & in biological properties but 100 times
more potent.
It is determined by filamentous phage integrated with the
bacterial chromosome, replicates as a plasmid and can
be transmitted to a nontoxigenic strain. Several genes
are involved in the virulence of V. cholerae O1-
Ctx A, Ctx B- for 2 subunits of cholera toxin,
Tcp- toxin corregulated pilus.
Gene complex,
acf- accessory colonization factor,
Hap- hemagglutination protease &
Neuraminidase
These genes are controlled by regulatory genes- Tox R.
Mechanism:- Molecular weight is 84,000, consist of one A and
five B subunits. B- subunit (binding)- binds to the ganglioside
GM1 receptors on the epithelial cells and promotes entry of
subunit A into the epithelial cells (jejunal).
A- subunit (active)- after entry divides into A1 & A2
fragments.
A2- Links the biologically active A1 to the B
subunit.
A1 fragments- enters the cell and activates
adenyl cyclase
- The enterotoxin causes the transfer of adenosine
diphosphate ribose (ADP ribose) from NAD (nicotinamide
adenine dinucleotide) to a regulatory protein, which is a part
of adenylate cyclase enzyme responsible for the generation of
intracellular cAMP (cyclic adenosine monophosphate).
This result into irreversible activation of adenylate cyclase and
overproduction of cAMP.
-It cause inhibition of uptake of Na+ & Cl- ions by cells lining
the villi and hyper secretion of Cl- & HCo3- ions, which blocks
uptake of water & passive outflow of water across mucosal
cells, leading to serious loss of water & electrolytes
-A1 fragment causes-
-Prolonged activation of adenylate cyclase & accumulation
of c-AMP leading to outpouring of large quantities of
water & electrolytes in lumen & the consequent watery
diarrhea.
-It is isotonic with plasma but contains much more of K+ &
HCo3-.
-All clinical manifestations & complications are due to
massive water & electrolyte depletion.
1 2
Mechanism
of Action of
Cholera
Toxin